The present invention relates to a modified compound, a method of chemoselective phosphorylation and a method of site-specific immobilization.
Chemoselective reactions have become an important tool in chemical research and in the biosciences, as they allow functional modules, for example biophysical probes, to be incorporated selectively in complex biomolecules. The essential element of chemoselective reactions is the selective transformation of a specific functionality within a complex biomolecule.
Various approaches are known for the selective functionalization of biomolecules, which are mainly included in the group of macromolecules. For example, naturally occurring functional groups such as thiols or phenols can be selectively modified, but site-specific labeling is not possible, as other thiols or phenols can also react.
Another approach comprises introducing nonnatural functionalities into biological molecules. Thus, it is possible, by established biochemical methods, to introduce azides into biological macromolecules, which are then available for further reaction. This method makes a site-specific modification possible, as the nonnatural functional group can be functionalized selectively by a chemoselective reaction.
Azide groups can for example be reacted chemoselectively with molecules with multiple bonds in the presence of copper(I) catalysts (Click Chemistry), but the use of toxic copper(I) catalysts is a disadvantage for application in living systems.
Another approach is described in WO 2001/068565 A2 or DE 601 20 650. Here, a chemoselective ligation reaction is carried out using phosphines for linking two reactants in mild reaction conditions (Staudinger ligation). This ligation reaction is based on the Staudinger reaction, in which an azide reacts chemoselectively with a phosphine to an iminophosphorane. To avoid hydrolysis of the iminophosphorane, this reaction can be manipulated so that an internal electrophilic substituent of the phosphine captures the resultant iminophosphorane, leading to formation of an amide bond in the end product. Drawbacks in this process are the slow reaction rate, the competitive iminophosphorane hydrolysis that still occurs, the sensitivity of phosphines to oxidation and the fact that the phosphines with the internal electrophilic substituent must be produced by a multistage process.